The present invention relates to transgenic plants on which capability of metabolizing, for example, drugs is conferred and use thereof.
Industrial development and advancement has increased kinds of environmental loads which destroy ecosystem. Moreover, numerous substances which disturb reproduction mechanism of organisms with an extremely small amount, such as extrinsic endocrine disruptors, have been reported. Environmental loads, extrinsic endocrine disruptors, and such are concentrated in the body of living organisms and more concentrated through food chain to profoundly affect living organisms and ecosystem. Therefore, it is necessary not only to remove these substances from environment, but also to metabolize and detoxify these in living organisms which have incorporated these substances.
Especially, plants are organisms belonging to the bottom of food chain, and plants in which these substances accumulate can be a source of destroying ecosystem. Thus, metabolizing and detoxifying these substances in plants are very important subjects.
Regarding agricultural crops which are closely related to our diet, agrochemicals such as herbicides, which are environmental loads, are generally used for cultivation. Thus, detoxifying residual agrochemicals in agricultural crops is needed for preventing adverse effect on humans and livestock which ingest these chemicals.
Cytochrome P450 monooxygenase (abbreviated to xe2x80x9cP450xe2x80x9d hereinafter) is an enzyme which distributes in a broad range of living organisms from microorganisms to plants and mammals and has a role of metabolizing and detoxifying drugs incorporated into living organisms. For P450 to exert its activity, another enzyme which provides p450 with electrons (abbreviated to xe2x80x9cP450 reductasexe2x80x9d hereinafter), for example, NADPH-P450 reductase, is required. This enzyme is also present in a broad spectrum of living organisms. The present inventors have created artificial enzymes showing extremely high oxidation activity by constructing fusion enzymegenes of various P450 molecular species and P450 reductases (Unexamined Published Japanese Patent Application (JP-A) No. Sho 63-44888, JP-A Hei 2-23870, and JP-A Hei 2-249488).
Recently, P450 in plants has been studied and various P450 molecular species broadly present in plants have been revealed. An activity of many drug-metabolizing P450 molecular species present in plants is, however, low, and capability of metabolizing environmental loads is extremely weak. Therefore, a novel plant with an enhanced capability of metabolizing environmental loads and such has been needed to create.
Specifically, it has been necessary to provide field crops which are used directly for foods and for feed and which play an important role in human life with the capability of metabolizing and detoxifying environmental loads and extrinsic endocrine disruptors represented by agrochemicals.
The present inventors have earnestly studied under these circumstances and successfully created transgenic plants on which capability of metabolizing, for example, drugs is conferred, by expressing drug-metabolizing P450 molecular species of mammals in plants. Transgenic plants of the present invention have the capability of metabolizing various herbicides depending on a molecular species of P450 expressed by them, and thus, show tolerance to these herbicides. Especially, an individual in which multiple P450 molecular species have been expressed can show higher tolerance to a broad spectrum of herbicides by the synergistic effect. Due to such a characteristic, the transgenic plants of the present invention are very useful as herbicide-tolerance plants. Moreover, these can absorb, metabolize, and decompose herbicides dispersed in the soil and, thus, are extremely environment-friendly. In addition, these plants can be used as a phytoremediation plant which can defecate environment by utilizing a plant (by using light energy) to metabolize and decompose not only herbicides but also environmental loads and extrinsic endocrine disruptors.
Therefore, the present invention provides transgenic plants which express a mono-oxygen addition activity derived from P450 or express a fusion enzyme activity comprising both mono-oxygen addition activity derived from P450 and reducing power-providing ability derived from P450 reductases and which can metabolize and decompose environmental loads and extrinsic endocrine disruptors, and use thereof.
Specifically, the present invention provides:
(1) a DNA molecule functionally comprising (a) a promoter capable of functioning in plant cells, (b) a P450 monooxygenase gene, and (c) a terminator capable of functioning in plant cells,
(2) the DNA molecule of (1), wherein the P450 monooxygenase gene is a fusion gene with a P450 reductase gene,
(3) the DNA molecule of (1) or (2), wherein the P450 monooxygenase gene is a gene belonging to CYP2 family,
(4) the DNA molecule of (3), wherein the gene belonging to CYP2 family is a gene selected from the group consisting of CYP2B6, CYP2C9, CYP2C18, and CYP2C19 genes,
(5) the DNA molecule of any one of (1) to (4), wherein the P450 monooxygenase gene is derived from mammals,
(6) a transgenic plant cell into which the DNA molecule of any one of (1) to (5) has been introduced,
(7) the transgenic plant cell of (6), wherein more than one type of P450 monooxygenase genes has been introduced into the transgenic plant cell,
(8) a transgenic plant comprising the plant cell of (6) or (7),
(9) the transgenic plant of (8), wherein the transgenic plant is a gramineous plant or a solanaceous plant,
(10) the transgenic plant of (8) or (9), wherein the transgenic plant is capable of decomposing a foreign compound by the oxidative metabolism,
(11) the transgenic plant of (10), wherein a foreign compound is an environmental load or an extrinsic endocrine disrupter,
(12) the transgenic plant of (10) or (11), wherein the transgenic plant comprises herbicide tolerance,
(13) a propagation material of the plant of any one of (8) to (12),
(14) a method for removing a foreign compound in environment, wherein the transgenic plant of (10) or (11) is used, and
(15) a method for selecting a P450 monooxygenase capable of metabolizing herbicides or a gene thereof, the method comprising:
(a) preparing a microsomal fraction from each transformant expressing one type of P450 monooxygenase gene introduced respectively into each transformant,
(b) reacting the microsomal fraction with an active ingredient of a herbicide,
(c) detecting a parent compound (the active ingredient of the herbicide) and/or a reaction product (a metabolite of the active ingredient of the herbicide), and
(d) selecting the P450 monooxygenase for which the reduction of the amount of the parent compound and/or the production of the reaction product has been detected, or a gene thereof.
In the present invention xe2x80x9cenvironmental loadsxe2x80x9d mean substances which are released to the environment and provide loads to the ecosystem. xe2x80x9cExtrinsic endocrine disruptorsxe2x80x9d mean substances which are present out of living organisms and cause hormone-like effect when incorporated into living organisms.
Any P450 molecular species can be used herein as long as they have a mono-oxygen addition activity, but those derived from mammals, which comprise higher activity than those derived from plants, are preferable. In this specification, xe2x80x9ca mono-oxygen addition activityxe2x80x9d means an activity catalyzing a reaction which adds a single oxygen atom to a substrate molecule. P450 molecular species belonging to CYP1, 2, 3, and 4 families are known to be mainly involved in the oxidative metabolism of foreign compounds, such as environmental loads including drugs, toxins, and agrochemicals, and thus, can be preferably used for the present invention (Imai, Y., and Kamataki, T., Protein, Nucleic acid and Enzyme, 1998, 43, 203-215). Specifically a preferable molecular species belonging to CYP1 family is derived from humans, for example, human CYP1A1 and human CYP1A2.
A herbicide metabolized by each P450 molecular species can be determined, for example, by preparing a microsomal fraction from a transformant in which each P450 molecule is expressed, by reacting the microsomal fraction with an active ingredient of each herbicide, and then, by detecting and analyzing a parent compound (an active ingredient of a herbicide) or a reaction product (a metabolite of the active ingredient in the herbicide).
Specifically, a P450 monooxygenase capable of metabolizing herbicides or the gene thereof can be selected by a method comprising:
(a) preparing a microsomal fraction from each transformant expressing one type of P450 monooxygenase gene introduced respectively into each transformant,
(b) reacting the microsomal fraction with an active ingredient of a herbicide,
(c) detecting a parent compound (the active ingredient of the herbicide) and/or a reaction product (a metabolite of the active ingredient of the herbicide), and
(d) selecting the P450 monooxygenase for which the reduction of the amount of the parent compound and/or the production of the reaction product has been detected, or the gene thereof.
Any types of P450 monooxygenase genes can be used for the selection as long as they have a mono-oxygen addition activity, but those derived from mammals are preferable because they can be expected to have higher herbicide-metabolizing ability compared with those derived from plants. A P450 mononxygenase gene can be introduced into, for example, yeast, insect cells, plant cells, and so on but not limited to them.
A microsomal fraction can be prepared by the various methods known to a person skilled in the art. An example is the method described in xe2x80x9cOeda, K. et al., DNA, 1985, 4, 203-210xe2x80x9d for yeast. Recombinant yeast strain microsomal fractions which express each molecular species of human P450 are available in the market (for example, Sumitomo Chemical).
Any herbicides can be used for a reaction. For example, the triazine compounds such as atrazine; the urea compounds such as chlorotoluron, diuron, and methabenzthiazuron; the diazine compounds such as norflurazon; the pyrimidinyloxybenzene compounds such as pyriminobac-methyl; the acetanilide compounds such as acetochlor, alachlor, and metolachlor; the dinitroaniline compounds such as triflurarin; the benzofuranylalkanesulfonate compounds such as benfuresate and ethofumesate; the sulfonylurea compunds such as chlorosulfuron and imazosulfuron; and the carbamate compounds such as pyributicarb can be used, but not limited thereto.
A microsomal fraction is reacted with an active ingredient of a herbicide by suspending the microsomal fraction in phosphate buffer at around pH 7 so that the amount of a P450 in the microsomal fraction is constant, and adding (i) NADPH for donating an electron to a P450 through a P450 reductase, (ii) a substrate and an enzyme for reproducing NADPH from NADP produced by donation of an electron (for example, glucose-6-phosphate and glucose-6-phosphate dehydrogenase), and (iii) a herbicide (a concentration depends on a type of herbicides). The reaction was carried out in a range of temperature suitable for an organism from which a p450 molecular species is derived (for example, 37xc2x0 C. for human P450), by mixing with air while vigorously shaking, within a period during which an enzyme is not inactivated (for example, for 10 to 60 min).
A parent compound (an active ingredient of a herbicide) or a reaction product (a metabolite of the active ingredient of the herbicide) can be detected and analyzed by, for example, the liquid chromatography (LC) and the liquid chromatography/mass spectrometry (LC/MS). If a peak of a parent compound decreases and instead a peak derived from a metabolite is confirmed, a sample used for an assay is determined to be capable of metabolizing a herbicide. From the height of the peak, a metabolized amount can be obtained.
The present inventors have been confirmed that, for example, human CYP1A1, human CYP2B6, human CYP2C18, human CYP2C19can metabolize respective herbicides below, and these can be preferably used for the present invention.
Human CYP1A1: N-deethylation, N-deisopropylation, and so on of the triazine compounds and such including atrazine; N-demethylation, ring hydroxylation, and so on of the urea compounds and such including chlorotoluron, diuron, methabenzthiazuron; N-demethylation and so on of the diazine compunds and such including norflurazon; hydroxylation and so on of the pyrimidinyloxybenzene compounds and such including pyriminobac-methyl.
Human CYP2B6: O-deethylation, O-demethylation, and so on of the acetanilide compounds and such including acetochlor, alachrol, metolachlor; O-demethylation and so on of the dinitroaniline compounds and such including triflurarin; O-deethylation, hydroxylation, and so on of the benzofuranylalkanesulfonate and such including benfuresate and ethofumesate.
Human CYP2C9: ring hydroxylation and so on of the sulfonylurea compounds and such including chlorosulfuron and imazosulfuron.
Human CYP2C18: N-demethylation, ring hydoxylation, and such of the urea compounds and such; O-deethylation, O-demethylation, and so on of the acetanilide compounds and such; O-demethylation and such of the dinitroaniline compounds and such; O-deethylation, hydroxylation, and so on of the benzofuranylalkanesulfonate compounds and such.
Human CYP2C19: N-deethylation, N-deisopropylation, and so on of the triazine compounds and such; N-demethylation, ring hydroxylation, and so on of the urea compounds and such; N-demethylation and so on of the diazine compounds and such; O-deethylation, O-demethylation, and so on of acetanilide compounds and such; thioester cleavage and so on of the carbamate compounds and such including pyributicarb; O-demethylation and so on of pyrimidinyloxybenzene compounds and such.
In the present invention, more than one type of P450 genes can be simultaneously expressed in a plant, and thereby, a capability of metabolizing a range of compounds broader than the case of expressing single P450 gene can be provided for a plant. Even when a plant does not show herbicide resistance due to its weak metabolizing capability from single P450 gene expression alone, herbicide tolerance can be provided for a plant by expressing more than one type of P450 genes to improve the metabolizing capability.
Any P450 reductases can be used in the present invention as long as they have a reducing power-providing activity to transfer an electron to a P450. For example, NADH-P450 reductases derived from microorganisms such as bacteria, NADPH-P450 reductases derived from mammals or plants, and so on can be used (Takemori, S., and Kominami, S., Cytochrome P-450, University of Tokyo Press).
In the present invention, a P450 can be solely expressed in a plant or with a P450 reductase which donates an electron to the P450. A P450 reductase can be expressed as a fusion protein with a P450. For constructing a fusion gene of a P450 gene and a P450 reductase gene, refer to the publications (JP-A Sho 63-44888, JP-A Hei 2-23870, and JP-A Hei 2-249488).
To express a gene in a plant cell, a DNA molecule (an expression cassette) functionally comprising (1) a promoter capable of functioning in plant cells, (2) the gene, and (3) a terminator capable of functioning in plant cells is prepared and introduced into the plant cells. Such a DNA molecule can contain a DNA sequence for improving transcription other than a promoter, for example, an enhancer sequence. Any promoters can be used as long as they function in plant cells. Examples are 35S (Schell, J. S., Science, 1987, 237, 1176-1183), Nos (Schell, J. S., Science, 1987, 237, 1176-1183), rbcS (Benefy, P. N., and N-H. Chua, Science, 1989, 244, 174-181), PR1a (Ohshima, M. et al., Plant Cell, 1990, 2, 95-106), ADH (Benefy, P. N. and N-H. Chua, Science, 1989, 244, 174-181), patatin (Benefy, P. N., and N-H. Chua, Science, 1989, 244, 174-181), Cab (Benefy, P. N., and N-H. Chua, Science, 1989, 244, 174-181), and PAL (Liang, X. et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 9284-9288). Such DNA molecules contain various vectors for expressing a gene.
A gene can be introduced into plant cells by the various methods known to a person skilled in the art. For example, indirect introduction methods using Agrobacterium tumefaciens or Agrobacterium rhizogenes (Hiei, Y. et al., Plant. J., 1994, 6, 271-282; Takaiwa, F. et al., Plant Sci., 1995, 111, 39-49) or direct introduction methods represented by electroporation method (Tada, Y. et al., Theor. Appl. Genet, 1990, 80, 475), a polyethylene glycol method (Datta, S. K., et al., Plant Mol Biol., 1992, 20, 619-629), and a particle gun method (Christou, P. et al., Plant J., 1992, 2, 275-281; Fromm, M. E., Bio/Technology, 1990, 8, 833-839) can be used. Any xe2x80x9cplant cellsxe2x80x9d can be used for gene introduction in the present invention as long as they are capable of regenerating an individual plant, and, for example, suspension cultured cells, calli, protoplasts, leaf slices, and so on are included.
An individual plant can be created by regenerating transformed plant cells. Plant species to be created in the present invention are not particularly limited, and, for example, gramineous plants such as rice, wheat, maize, gramineous pasture, and turf; solaneceous plants such as tobacco, tomato, and potato; crucifer crops such as rape; and plants such as sunflower and alfalfa, are especially preferable as a target plant on which capability of metabolizing drugs and such is conferred. Methods for regenerating an individual plant depend on a species of plant cells. Representative methods are, for example, the method by Fujimura et al. for rice (Fujimura, T. et al., Plant Tissue Culture. Lett., 1995, 2, 74), the methods described in the references (Akbar, S. et al., Plant Cell, Tissue and Organ Culture, 1991, 26, 185-187; Altpeter, F. et al., Plant Cell Rep., 1996, 16, 12-17) for wheat, the methods described in the references (Singh, R. R., et al., Plant Cell, Tissue and Organ Culture, 1997, 49, 121-127) for barley, the method described by Armstrong et al. (Armstrong, C. L. and Phillips, R. L., Crop Sci. , 1988, 28, 363-369) for maize, the method described in the reference (Dalton, S. J., Biotechnology in Agriculture and Forestry, 1993, 22, 46-68) for Italian rye grass, the method described in the reference (Asano, Y., Plant Cell Rep., 1989, 8, 141-143) for turf, the method described in the reference (Rogers, S. G. et al., Methods Enzymol., 1986, 118, 627-640) for tobacco, the method described in the reference (Sheerman, S and Bevan, M. W., Plant Cell Rep., 1988, 7, 13-16) for potato, the method described in the reference (Kohno-Murase, J. et al., Plant Mol. Biol., 1994, 26, 1115-1124) for rape, the method described in the reference (Schrammeijer, B. et al., Plant Cell Rep., 1990, 9, 55-60) for sunflower, and the method described in the reference (Shahin, E. A. et al., Crop Sci., 1986, 26, 1235-1239) for alfalfa.
An individual plant created in the present invention or that created from propagation materials (for example, seeds, tuberous roots, tubers, fruits, ear, etc.) thereof is capable of oxidatively metabolizing and detoxifying foreign compounds, for example, environmental loads and extrinsic endocrine disruptors including drugs, toxins, and agrochemicals.
More tolerant individuals can be selected by examining tolerance to one or more herbicides in the obtained transgenic plants. Preferably, the function of an introduced gene in a selected individual is confirmed by assaying introduction of a target P450 cDNA into chromosomal DNA, production of mRNA, production of a P450 protein corresponding to the introduced gene, a P450 enzyme activity, etc.
Specific examples of drugs and toxins to be oxidatively metabolized and detoxified by the individual plants of the present invention are, for example, benzopyrene and such for CYP1A1 (Shimada, T. et al., Cancer Res., 1991, 49, 6304-6312), nicotine and such for CYP2B6 (Flammang, A. M. et al., Biochem. Arch., 1992, 8, 1-8), Ibuprofen (Leeman, T. et al., 1993, Life Sci., 52: 29-34), Tolbutamide (Brian, W. R. et al., 1989, Biochemistry, 28: 4993-4999), and such for CYP2C9, Tolbutamide and such for CYP2C18 (Furuya, H., Mol. Pharmacol. 1991, 40, 375-382), Mephenytoin (Shimada, T. et al., 1986, J. Biol. Chem., 261: 909-921), Omeprazol (Anderson T. et al., 1990, Ther. Drug Monitoring, 12: 415-416), and such for CYP2C19, but not limited thereto.
Examples of agrochemicals are the acetanilide or dinitroaniline herbicides and the like, such as alachlor (Monsanto etc.), metolachlor (former Ciba-Geigy, Novartis, etc.), triflurarin (Eli Lilly etc.), and acetochlor (Monsanto etc.); the sulfonylurea herbicides and the like, such as chlorosulfuron (E. I. du pont de Nemous and Co. Inc. etc.) and imazosulfuron (Takeda Chemical industries, Ltd., etc.); the carbamate herbicides and the like, such as pyributicarb (Toso, etc.); the triazine herbicides and the like, such as simazine (former Chiba-Geigy, Novartis, etc.) and atrazine (former Geigy, Novartis, etc.); the diazine herbicide and the like, such as norflurazon (former Santos, Novartis, etc.); the urea herbicides and the like, such as chlorotoluron (former Chiba, Novartis, etc.) and methabenzthiazuron (Bayer etc.); but not limited thereto.
Therefore, environmental loads can be reduced or tolerance to the compounds can be provided by directly or indirectly contacting the plants of the present invention with these foreign compounds. Moreover, accumulation of the foreign compounds in vivo or residual agrochemicals can be reduced.
In developing countries and such, appropriate water management is difficult in most paddy fields, and control of weeds is cumbersome in these paddies. Rice plants capable of metabolizing drugs created in the present invention acquire resistance to herbicides by metabolizing the herbicides. Cultivating these rice plants makes control of weeds efficient without increasing loads to environmental. Similarly, these rice plants are effective for controlling weeds at the early stage in direct sowing fields.
In addition, parasitic plants such as Orobanche (Japanese name Hamautsubo) and Cuscuta (Japanese name Hamanenashikazura) parasitize various crops such as tobacco, tomato, potato, sunflower, and alfalfa. So far, there is no herbicide which can distinguish between parasitic plants and host plants. By introducing P450 genes such as CYP2C9, into hosts, parasitic plants can be controlled by a small amount of drugs.